PROJECT SUMMARY The current 3D genome mapping technologies (including ChIA-PET and Hi-C) require millions of cells for each assay and primarily rely on proximity ligation, PCR amplification of the ligation products, and Illumina-based high throughput sequencing. While such methods have revealed many principles of chromatin interaction and useful 3D genome organizational maps based on signals averaged over millions of cells, they provided only binary (paired) chromatin contact information, and thus do not enable elucidating the precise molecular mechanisms of complex chromatin interactions occurring in individual cells. The development of single-cell 3D genome mapping technologies has thus attracted significant interest in the research community. However, current single cell strategies are still largely based on the conventional molecular approaches for DNA manipulation mentioned above, which present technical barriers that prevent true single-molecule and single cell analyses genome-wide. In this project, we propose to develop a completely new system of next generation technology for chromatin interaction assays based on the SeqLL single-molecule protein-detection and DNA-sequencing platform. A prototype single-molecule platform has been demonstrated for simultaneous detection of histone modifications and genomic positions of individual nucleosomes (Shema et al., 2016). We will adapt this platform to enable single-molecule Chromatin Interaction Analysis (smChIA) in single nuclei. The proposed smChIA method possesses several important advantages over the current methods, including single-molecule resolution in single nuclei, no need of PCR or any amplification, and most significantly the elimination of proximity ligation steps. To aggressively advance the development of this next generation chromatin interaction analysis technology, we have assembled an integrative team with interdisciplinary and complementary knowledge and skills to ensure successful implementation of the specific aims in the proposal. We have also established an academic-industry partnership to make quick dissemination of the technology to the larger research community. Our preliminary results have provided initial proof-of-concept for the strategy of smChIA. We believe smChIA has the potential to revolutionize the entire field of 3D genome biology from data acquisition to interpretation. Once fully developed, we expect that the smChIA data will generate exciting and novel biological insights in the organization of the 3D genome and its dynamic modulation.